Increased Activity of the S Phase Kinase Cdc7 Is Associated with Poor Outcome in Diffuse Large B Cell Lymphoma (DLBCL).

Abnormal proliferation is associated with poor outcome in patients with diffuse large B cell lymphoma (DLBCL), with Ki-67 a well established prognostic marker. More recently gene expression profiling has shown high MYC expression, a marker of proliferation, to be associated with poor outcome in DLBCL. Thus, the cell cycle is an attractive therapeutic target in aggressive lymphoma. Cdc7, a serine/threonine kinase is essential for initiation of DNA replication and cell cycle regulation. Cdc7 phosphorylation of the replicative helicase Mcm2 is a key event in this process. Overexpression of Mcm2 has been linked to increased proliferation and poor outcome in a variety of cancers, including DLBCL. No data exists on the prognostic relevance of the phosphorylated form of Mcm2. Recently, overexpression of Cdc7 and its regulatory subunit Dbf4 has been demonstrated in multiple cancers but again, the prognostic relevance of this is unknown. Emerging data shows that inhibition of Cdc7 activity with a new class of pyrrolopyridinone kinase inhibitors has activity against a broad range of cancers, including lymphoma cell lines, even in cells lacking p53.

We hypothesized that increased Cdc7 expression and/or Mcm2 phosphorylation may be associated with poor prognosis in diffuse large B cell lymphoma (DLBCL) and that if confirmed, Cdc7 inhibition could be a potential treatment strategy in DLBCL.

The primary aim of this project was to explore the prognostic significance of Mcm2 phosphorylation and Cdc7 expression in archived diagnostic biopsy material from patients with DLBCL.

All patients with a confirmed diagnosis of treated for DLBCL treated in Galway University Hospital between 2001 and 2009 and having suitable material were selected. Chart review was undertaken to document baseline demographics, International Prognostic Index (IPI), treatment, remission rate, progression free survival (PFS) and overall survival (OS). Two tissue cores (0.6mm diameter) from each specimen were arrayed onto a tissue microarray (TMA). Expression of phospho-Mcm2 and Cdc7 as well as Ki-67, p53, Mcm2, c-myc, standard germinal centre and non-germinal centre markers were evaluated by immunohistochemisty (IHC) on the TMAs. Expression of these markers was evaluated against clinical endpoints. PFS and OS curves were constructed using the method of Kaplan and Meier and evaluated using the log-rank test. The relationship between phospho-Mcm2, Cdc7 and the known prognostic factors: germinal center and non-germinal centre type, Ki-67, and c-myc were explored through Mann-Whitney rank test.

Seventy-eight patients including 43 males (median age 62) and 35 females (median age 67) were included. 10% of patients were in the low, 37% in the low-intermediate, 24% in the high —intermediate and 28% in the high IPI group. IPI score was significantly associated with survival (p<0.05), with median OS 52 months in low, 32 in low-intermediate, 40 in high —intermediate and 15 months in high IPI group. Median observation time was 28 months and 57 patients had received rituximab based therapies. The median Cdc7 expression score was 5%, (0-50%). A value >10% was set as the cut off for high Cdc7. The cumulative OS at 50 months was 57% in the low Cdc7 group and 37% in the high Cdc7 group (p=0.26) for the whole cohort. For the patients with a low and low-intermediate IPI the cumulative OS at 50 months was 68% in the low Cdc7 group and 40% in the high Cdc7 group (p=0.1). For patients with a high and intermediate-high IPI, the cumulative OS at 50 months was 51% in low Cdc7 group and 42 % in high Cdc7 group (p=0.9). The PFS was not significantly different in the whole cohort as well as in IPI subgroups between patient with high and low Cdc7. Interestingly there was a significant correlation between Cdc7 and Ki67 expression (p<0.05) . Cdc7 expression was not correlated with CD10, bcl6, bcl2 or germinal centre versus non-germinal centre. The p-Mcm2, Dbf4, c-myc and p53 staining is ongoing and the results will be presented.

Despite a relatively small sample size, our results suggest that increased activity of the Cdc7 kinase may be associated with a trend towards worse outcome. This is particularly apparent in low to low-intermediate IPI. Cdc7 could be also a potential therapeutic target in DLBCL. Further studies are needed to characterise prognostic and therapeutic significance of high expression of Cdc7 and pMcm2.

No relevant conflicts of interest to declare.